Clonality analysis by T-cell receptor gamma PCR and high-resolution electrophoresis in the diagnosis of cutaneous T-cell lymphoma (CTCL).

Abstract

During T-cell maturation, T-cell receptor (TCR) gene segments rearrange, resulting in a new, unique DNA configuration. The recombined TCR gene loci display a high degree of nucleotide sequence variability. Molecular biological clonality assays focus on this cell-specific DNA pattern. The finding of an identical TCR rearrangement in a large number of T lymphocytes signals a malignant proliferation, although clonality is not always equivalent to malignancy. Thus, detection of clonal TCR gamma rearrangements by polymerase chain reaction (PCR), followed by high-resolution electrophoresis is a valuable tool in the diagnosis of cutaneous and other T-cell lymphomas. For the clonality assay described here, all rearrangements of T cells present in a given sample are amplified by a set of only three TCR gamma-PCRs. The products are investigated by either heteroduplex temperature gradient gel electrophoresis (HD-TGGE) or fluorescent fragment analysis (FFA) on a capillary DNA sequencer (or by both methods), for clonality. Both electrophoresis techniques show highly reproducible results and are comparatively easy to conduct, however, specific instruments are required. Concerning lower detection thresholds, the methods need a minimum of about 1% of clonal T-cells in mixtures with polyclonal T-cells for revealing clonality.